Enterohemorrhagic E. coli (EHEC) is a significant public health problem because of the serious nature of the sequelae that can result from the infection. EHEC infection can progress from bloody diarrhea to serious kidney disease, HUS, and even death. Shiga toxin (Stx), a major virulence factor in EHEC and the cause of HUS, is usually carried in the genome of bacterial viruses called phages. Because these phages grow either lytically, releasing progeny into the environment, or lysogenically, inserting their DNA into the bacterial genome (as prophages), they are important agents for horizontal transfer of stx genes. In a fraction of lysogens, the prophage switches to lyric growth through a process called induction. Our work has shown that induced prophages add to the infectious process through their roles in increasing gene copy number of stx and through action of their regulatory systems contributing to the expression and release of Stx. The experiments outlined in this proposal are designed to examine the process of induction of Stx phage 933W and its role in Stx production and how the intestinal environment contributes to this process. We have constructed a bacterial reporter system that provides a genetic mechanism for identifying those bacteria in which the prophage is induced causing expression of phage-encoded genes, including stx. We plan to make improvements in this system, transfer it to an EHEC strain, and use the constructed strain to study how the in vivo environment contributes prophage induction and, thus, to Stx expression and release. We have shown that phage 933W encodes a functional eukaryotic-like tyrosine kinase, Stk, that is expressed in EHEC strains that have 933W prophages. We propose experiments designed to determine if Stk contributes to the virulence of EHEC strains. These studies should provide new and important information on factors influencing the course of EHEC infections. Moreover, this information should also contribute in a general way to the understanding of how interactions of bacteria and their environment influence the activity of their prophages, many of which encode virulence factors. [unreadable] [unreadable]